Photonic Molecule Approach to Multiorbital Topology.

The concepts of topology provide a powerful tool to tailor the propagation and localization of the waves. While electrons have only two available spin states, engineered degeneracies of photonic modes provide novel opportunities resembling spin degrees of freedom in condensed matter. Here, we tailor such degeneracies for the array of femtosecond laser written waveguides in the optical range exploiting the idea of photonic molecules: clusters of strongly coupled waveguides. In our experiments, we observe unconventional topological modes protected by the Z3 invariant arising due to the interplay of interorbital coupling and geometric dimerization mechanism. We track multiple topological transitions in the system with the change in the lattice spacings and excitation wavelength. This strategy opens an avenue for designing novel types of photonic topological phases and states.

IVEN: A quantitative tool to describe 3D cell position and neighbourhood reveals architectural changes in FGF4-treated preimplantation embryos.

Architectural changes at the cellular and organism level are integral and necessary to successful development and growth. During mammalian preimplantation development, cells reduce in size and the architecture of the embryo changes significantly. Such changes must be coordinated correctly to ensure continued development of the embryo and, ultimately, a successful pregnancy. However, the nature of such transformations is poorly defined during mammalian preimplantation development. In order to quantitatively describe changes in cell environment and organism architecture, we designed Internal Versus External Neighbourhood (IVEN). IVEN is a user-interactive, open-source pipeline that classifies cells into different populations based on their position and quantifies the number of neighbours of every cell within a dataset in a 3D environment. Through IVEN-driven analyses, we show how transformations in cell environment, defined here as changes in cell neighbourhood, are related to changes in embryo geometry and major developmental events during preimplantation mammalian development. Moreover, we demonstrate that modulation of the FGF pathway alters spatial relations of inner cells and neighbourhood distributions, leading to overall changes in embryo architecture. In conjunction with IVEN-driven analyses, we uncover differences in the dynamic of cell size changes over the preimplantation period and determine that cells within the mammalian embryo initiate growth phase only at the time of implantation.

Synthesis of a new 2-prenylated quinoline as potential drug for metabolic syndrome with pan-PPAR activity and anti-inflammatory effects.

We have previously reported the total synthesis and structure-activity relationships (SAR) of 2-prenylated benzopyrans with PPAR agonist activity. Herein, we have described the synthesis and PPAR activity of 2-prenylated benzopyrans and 2-prenylated quinolines. The benzopyran nucleus was generated via enamine-catalyzed Kabbe condensation, and the quinoline nucleus via Friedländer condensation. Results demonstrated that both benzopyran (5a) and quinoline (4b) derivatives bearing a γ,δ-unsaturated ester displayed a pan-PPAR agonism. They were full PPARα agonists, but showed different preferences for PPARγ and PPARß/δ activation. It was noteworthy that quinoline 4b displayed full hPPARα activation (2-fold than WY-14,643), weak PPARß/δ and partial PPARγ activation. In addition, quinoline 4b showed anti-inflammatory effects on macrophages by reducing LPS-induced expression of both MCP-1 and IL-6. Therefore, 4b emerges as a first-in-class promising hit compound for the development of potential therapeutics aimed at treating metabolic syndrome, metabolic dysfunction-associated fatty liver disease (MAFLD), and its associated cardiovascular comorbidities.

Daam2 couples translocation and clustering of Wnt receptor signalosomes through Rac1.

Wnt signaling plays a critical role in development across species and is dysregulated in a host of human diseases. A key step in signal transduction is the formation of Wnt receptor signalosomes, during which a large number of components translocate to the membrane, cluster together and amplify downstream signaling. However, the molecular processes that coordinate these events remain poorly defined. Here, we show that Daam2 regulates canonical Wnt signaling via the PIP2-PIP5K axis through its association with Rac1. Clustering of Daam2-mediated Wnt receptor complexes requires both Rac1 and PIP5K, and PIP5K promotes membrane localization of these complexes in a Rac1-dependent manner. Importantly, the localization of Daam2 complexes and Daam2-mediated canonical Wnt signaling is dependent upon actin polymerization. These studies - in chick spinal cord and human and monkey cell lines - highlight novel roles for Rac1 and the actin cytoskeleton in the regulation of canonical Wnt signaling and define Daam2 as a key scaffolding hub that coordinates membrane translocation and signalosome clustering.

Anti-inflammatory effects and improved metabolic derangements in ob/ob mice by a newly synthesized prenylated benzopyran with pan-PPAR activity.

BACKGROUND AND PURPOSE: Selective peroxisome proliferator-activated receptors (PPARs) are widely used to treat metabolic complications; however, the limited effect of PPARα agonists on glucose metabolism and the adverse effects associated with selective PPARγ activators have stimulated the development of novel pan-PPAR agonists to treat metabolic disorders. Here, we synthesized a new prenylated benzopyran (BP-2) and evaluated its PPAR-activating properties, anti-inflammatory effects and impact on metabolic derangements. EXPERIMENTAL APPROACH: BP-2 was used in transactivation assays to evaluate its agonism to PPARα, PPARß/δ and PPARγ. A parallel-plate flow chamber was employed to investigate its effect on TNFα-induced leukocyte-endothelium interactions. Flow cytometry and immunofluorescence were used to determine its effects on the expression of endothelial cell adhesion molecules (CAMs) and chemokines and p38-MAPK/NF-κB activation. PPARs/RXRα interactions were determined using a gene silencing approach. Analysis of its impact on metabolic abnormalities and inflammation was performed in ob/ob mice. KEY RESULTS: BP-2 displayed strong PPARα activity, with moderate and weak activity against PPARß/δ and PPARγ, respectively. In vitro, BP-2 reduced TNFα-induced endothelial ICAM-1, VCAM-1 and fractalkine/CX3CL1 expression, suppressed mononuclear cell arrest via PPARß/δ-RXRα interactions and decreased p38-MAPK/NF-κB activation. In vivo, BP-2 improved the circulating levels of glucose and triglycerides in ob/ob mice, suppressed T-lymphocyte/macrophage infiltration and proinflammatory markers in the liver and white adipose tissue, but increased the expression of the M2-like macrophage marker CD206. CONCLUSION AND IMPLICATIONS: BP-2 emerges as a novel pan-PPAR lead candidate to normalize glycemia/triglyceridemia and minimize inflammation in metabolic disorders, likely preventing the development of further cardiovascular complications.

Regional heterogeneity of astrocyte morphogenesis dictated by the formin protein, Daam2, modifies circuit function.

Astrocytes display extraordinary morphological complexity that is essential to support brain circuit development and function. Formin proteins are key regulators of the cytoskeleton; however, their role in astrocyte morphogenesis across diverse brain regions and neural circuits is unknown. Here, we show that loss of the formin protein Daam2 in astrocytes increases morphological complexity in the cortex and olfactory bulb, but elicits opposing effects on astrocytic calcium dynamics. These differential physiological effects result in increased excitatory synaptic activity in the cortex and increased inhibitory synaptic activity in the olfactory bulb, leading to altered olfactory behaviors. Proteomic profiling and immunoprecipitation experiments identify Slc4a4 as a binding partner of Daam2 in the cortex, and combined deletion of Daam2 and Slc4a4 restores the morphological alterations seen in Daam2 mutants. Our results reveal new mechanisms regulating astrocyte morphology and show that congruent changes in astrocyte morphology can differentially influence circuit function.

Multiplex Hybrid Antigen-Capture LC-MRM Quantification in Sera and Nasal Lining Fluid of AZD7442, a SARS-CoV-2-Targeting Antibody Combination.

AZD7442 (tixagevimab [AZD8895]/cilgavimab [AZD1061]) is a monoclonal antibody (mAb) combination in development for the prevention and treatment of coronavirus disease 2019. Traditionally, bioanalysis of mAbs is performed using ligand binding assays (LBAs), which offer sensitivity, robustness, and ease of implementation. However, LBAs frequently require generation of critical reagents that typically take several months. Instead, we developed a highly sensitive (5 ng/mL limit of quantification) method using a hybrid LBA-liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) approach for quantification of the two codosed antibodies in serum and nasal lining fluid (NLF), a rare matrix. The method was optimized by careful selection of multiple reaction monitoring, capture reagents, magnetic beads, chromatographic conditions, evaluations of selectivity, and matrix effect. The final assay used viral spike protein receptor-binding domain as capture reagent and signature proteotypic peptides from the complementarity-determining region of each mAb for detection. In contrast to other methods of similar/superior sensitivity, our approach did not require multidimensional separations and can be operated in an analytical flow regime, ensuring high throughput and robustness required for clinical analysis at scale. The sensitivity of this method significantly exceeds typical sensitivity of ∼100 ng/mL for analytical flow 1D LBA-LC-MS/MS methods for large macromolecules, such as antibodies. Furthermore, infection and vaccination status did not impact method performance, ensuring method robustness and applicability to a broad patient population. This report demonstrated the general applicability of the hybrid LBA-LC-MS/MS approach to platform quantification of antibodies with high sensitivity and reproducibility, with specialized extension to matrices of increasing interest, such as NLF.

Synthesis and biological studies of "Polycerasoidol" and "trans-δ-Tocotrienolic acid" derivatives as PPARα and/or PPARγ agonists.

2-Prenylated benzopyrans represent a class of natural and synthetic compounds showing a wide range of significant activities. Polycerasoidol is a natural prenylated benzopyran isolated from the stem bark of Polyalthia cerasoides (Annonaceae) that exhibits dual PPARα/γ agonism and an anti-inflammatory effect by inhibiting mononuclear leukocyte adhesion to the dysfunctional endothelium. Herein, we report the synthesis of three new series of prenylated benzopyrans containing one (series 1), two (series 2, "polycerasoidol" analogs) and three (series 3, "trans-δ-tocotrienolic acid" analogs) isoprenoid units in the hydrocarbon side chain at the 2-position of the chroman-6-ol (6-hydroxy-dihydrobenzopyran) scaffold. Isoprenoid moieties were introduced through a Grignard reaction sequence, followed by Johnson-Claisen rearrangement and subsequent Wittig olefination. hPPAR transactivation activity and the structure activity relationships (SAR) of eleven novel synthesized 2-prenylated benzopyrans were explored. PPAR transactivation activity demonstrated that the seven-carbon side chain analogs (series 1) displayed selectivity for hPPARα, while the nine-carbon side chain analogs (polycerasoidol analogs, series 2) did so for hPPARγ. The side chain elongation to 11 or 13 carbons (series 3) resulted in weak dual PPARα/γ activation. Therefore, 2-prenylated benzopyrans of seven- and nine-carbon side chain (polycerasoidol analogs) are good lead compounds for developing useful candidates to prevent cardiovascular diseases associated with metabolic disorders.

Regulation of Pv-specific interneurons in the medial prefrontal cortex and reward-seeking behaviors.

The corticostriatal circuitry and its glutamate-γ-aminobuturic acid (GABA) interactions play an essential role in regulating neuronal excitability during reward-seeking behavior. However, the contribution of GABAergic interneurons in the corticostriatal circuitry remains unclear. To investigate the role of GABAergic interneurons, we focused on parvalbumin-expressing fast-spiking interneurons (Pv-FSI) in the corticostriatal circuitry using the designer receptors exclusively activated by designer drugs approach in a Pv-Cre mouse model. We hypothesize that Pv-FSI activation elicits changes in cortical glutamate levels and reward-seeking behaviors. To determine molecular and behavioral effects of Pv-FSI, we performed microdialysis and operant conditioning tasks for sucrose and alcohol rewards. In addition, we also examined how alcohol reward itself affects Pv-FSI functioning. Interestingly, our microdialysis results demonstrate that alcohol exposure inhibits Pv-FSI functioning in the medial prefrontal cortex (mPFC) and this consequently can regulate glutamate levels downstream in the nucleus accumbens. For sucrose reward-seeking behaviors, Pv-FSI activation in the mPFC increases sucrose self-administration whereas it does not promote alcohol seeking. For alcohol rewards, however, Pv-FSI activation in the mPFC results in increased compulsive head entry in operant chambers during devaluation procedures. Overall, our results suggest that not only do Pv-FSI contribute to changes in the cortical microcircuit and reward-seeking behaviors but also that alcohol affects Pv-FSI neurotransmission. Therefore, Pv-FSI has prompted interest in their role in maintaining a balance in neuronal excitation/inhibition and in regulating reward-seeking processes such as compulsivity, all of which are important factors for excessive alcohol seeking.

Multifaceted Bioanalytical Methods for the Comprehensive Pharmacokinetic and Catabolic Assessment of MEDI3726, an Anti-Prostate-Specific Membrane Antigen Pyrrolobenzodiazepine Antibody-Drug Conjugate.

Complex biotherapeutic modalities, such as antibody-drug conjugates (ADC), present significant challenges for the comprehensive bioanalytical characterization of their pharmacokinetics (PK) and catabolism in both preclinical and clinical settings. Thus, the bioanalytical strategy for ADCs must be designed to address the specific structural elements of the protein scaffold, linker, and warhead. A typical bioanalytical strategy for ADCs involves quantification of the Total ADC, Total IgG, and Free Warhead concentrations. Herein, we present bioanalytical characterization of the PK and catabolism of a novel ADC. MEDI3726 targets prostate-specific membrane antigen (PMSA) and is comprised of a humanized IgG1 antibody site-specifically conjugated to tesirine (SG3249). The MEDI3726 protein scaffold lacks interchain disulfide bonds and has an average drug to antibody ratio (DAR) of 2. Based on the structural characteristics of MEDI3726, an array of 4 bioanalytical assays detecting 6 different surrogate analyte classes representing at least 14 unique species was developed, validated, and employed in support of a first-in-human clinical trial (NCT02991911). MEDI3726 requires the combination of heavy-light chain structure and conjugated warhead to selectively deliver the warhead to the target cells. Therefore, both heavy-light chain dissociation and the deconjugation of the warhead will affect the activity of MEDI3726. The concentration-time profiles of subjects dosed with MEDI3726 revealed catabolism of the protein scaffold manifested by the more rapid clearance of the Active ADC, while exhibiting minimal deconjugation of the pyrrolobenzodiazepine (PBD) warhead (SG3199).

1-(2'-Bromobenzyl)-6,7-dihydroxy-N-methyl-tetrahydroisoquinoline and 1,2-Demethyl-nuciferine as Agonists in Human D2 Dopamine Receptors.

Certain D2-like dopamine receptor (DR) agonists are useful therapeutically as antiparkinsonian drugs, whereas D2-like DR antagonists or partial agonists are proven effective as antipsychotics. Two isoquinoline derivatives, 1-(2'-bromobenzyl)-6,7-dihydroxy-N-methyl-tetrahydroisoquinoline (Br-BTHIQ, 1) and 1,2-demethyl-nuciferine (aporphine, 2), were herein synthesized, and their dopaminergic affinity in cloned human D2R, D3R, and D4R subtypes and their behavior as agonists/antagonists were evaluated. They showed affinity values (Ki) for hD2, hD3, and hD4 DR within the nanomolar range. The trends in affinity were hD4R ≫ hD3R > hD2R for Br-BTHIQ (1) and hD2R > hD4R > hD3R for 1,2-demethyl-nuciferine (2). The functional assays of cyclic adenosine monophosphate signaling at human D2R showed a partial agonist effect for Br-BTHIQ (1) and full agonist behavior for aporphine (2), with half maximal effective concentration values of 2.95 and 10.2 µM, respectively. Therefore, both isoquinolines 1 and 2 have emerged as lead molecules for the synthesis of new therapeutic drugs that ultimately may be useful to prevent schizophrenia and Parkinson's disease, respectively.

Synthesis of benzopyran derivatives as PPARα and/or PPARγ activators.

We describe the synthesis of 26 compounds, small polycerasoidol analogs, that are Lipinski's rule-of-five compliant. In order to confirm key structural features to activate PPARα and/or PPARγ, we have adopted structural modifications in the following parts: (i) the benzopyran core (hydrophobic nucleus) by benzopyran-4-one, dihydrobenzopyran or benzopyran-4-ol; (ii) the side chain at 2-position by shortening to C3, C4 and C5-carbons versus C-9-carbons of polycerasoidol; (iii) the carboxylic group (polar head) by oxygenated groups (hydroxyl, acetoxy, epoxide, ester, aldehyde) or non-oxygenated motifs (allyl and alkyl). Benzopyran-4-ones 6, 12, 13 and 17 as well as dihydrobenzopyrans 22, 24 and 25 were able to activate hPPARα, whereas benzopyran-4-one (7) with C5-carbons in the side chain exhibited hPPARγ agonism. According to our previous docking studies, SAR confirm that the hydrophobic nucleus (benzopyran-4-one or dihydrobenzopyran) is essential to activate PPARα and/or PPARγ, and the flexible linker (side alkyl chain) should containg at least C5-carbon atoms to activate PPARγ. By contrast, the polar head ("carboxylic group") tolerated several oxygenated groups but also non-oxygenated motifs. Taking into account these key structural features, small polycerasoidol analogs might provide potential active molecules useful in the treatment of dyslipidemia and/or type 2 diabetes.

Polycerasoidol, a Natural Prenylated Benzopyran with a Dual PPARα/PPARγ Agonist Activity and Anti-inflammatory Effect.

Dual peroxisome proliferator-activated receptor-α/γ (PPARα/γ) agonists regulate both lipid and glucose homeostasis under different metabolic conditions and can exert anti-inflammatory activity. We investigated the potential dual PPARα/γ agonism of prenylated benzopyrans polycerasoidol (1) and polycerasoidin (2) and their derivatives for novel drug development. Nine semisynthetic derivatives were prepared from the natural polycerasoidol (1) and polycerasoidin (2), which were evaluated for PPARα, -γ, -δ and retinoid X receptor-α activity in transactivation assays. Polycerasoidol (1) exhibited potent dual PPARα/γ agonism and low cytotoxicity. Structure-activity relationship studies revealed that a free phenol group at C-6 and a carboxylic acid at C-9' were key features for dual PPARα/γ agonism activity. Molecular modeling indicated the relevance of these groups for optimal ligand binding to the PPARα and PPARγ domains. In addition, polycerasoidol (1) exhibited a potent anti-inflammatory effect by inhibiting mononuclear leukocyte adhesion to the dysfunctional endothelium in a concentration-dependent manner via RXRα/PPARγ interactions. Therefore, polycerasoidol (1) can be considered a hit-to-lead molecule for the further development of novel dual PPARα/γ agonists capable of preventing cardiovascular events associated with metabolic disorders.

Kinetics of the Aqueous Phase Reactions of Atmospherically Relevant Monoterpene Epoxides.

Laboratory and field measurements have demonstrated that an isoprene-derived epoxide intermediate (IEPOX) is the origin of a wide range of chemical species found in ambient secondary organic aerosol (SOA). In order to explore the potential relevance of a similar mechanism for the formation of monoterpene-derived SOA, nuclear magnetic resonance techniques were used to study kinetics and reaction products of the aqueous-phase reactions of several monoterpene epoxides: ß-pinene oxide, limonene oxide, and limonene dioxide. The present results, combined with a previous study of α-pinene oxide, indicate that all of these epoxides will react more quickly than IEPOX with aqueous atmospheric particles, even under low-acidity conditions. As for α-pinene oxide, the observed products can be mainly rationalized with a hydrolysis mechanism, and no long-lived organosulfate or nitrate species nor species that retain the ß-pinene bicyclic carbon backbone are observed. As bicyclic ring-retaining organosulfate and nitrate species have been previously observed in monoterpene-derived SOA, it appears that monoterpene-derived epoxides may not be as versatile as IEPOX in producing a range of SOA species, and other mechanisms are needed to rationalize organosulfate and nitrate formation.

Gas Phase Oxidation of Campholenic Aldehyde and Solution Phase Reactivity of its Epoxide Derivative.

The rate constant for the OH reaction with campholenic aldehyde (CA) was measured using the flow tube-chemical ionization mass spectrometry method with a relative rate kinetics technique and was found to be (6.54 ± 0.52) × 10-11 cm3 molecule-1 s-1 at 100 Torr pressure and 298 K. A mechanism for the formation of the observed products was developed for both NO-free and NO-present conditions. On the basis of measurements of the pressure dependent yields of the products, between 5 and 20% of the CA oxidation at atmospheric pressure is predicted to lead to campholenic aldehyde epoxide (CAE). The aqueous solution reaction rate constants for CAE were determined via NMR spectroscopy and were found to be (2.241 ± 0.036) × 10-5 s-1 for neutral conditions and 0.0989 ± 0.0053 M-1 s-1 for acid-catalyzed conditions at 298 K. The products of the CAE aqueous solution reaction were identified as an isomer of CAE and the aldehyde group hydrated form of this isomer. Unlike the isoprene-derived epoxide, IEPOX, a nucleophilic addition mechanism was not observed. On the basis of the rate constants determined for CA and CAE, it is likely that these species are reactive on atmospherically relevant time scales in the gas and aerosol phases, respectively. The results of the present study largely support a previous supposition that α-pinene-derived secondary organic aerosol may be influenced by the multiphase processing of various intermediate species, including those with epoxide functionality.

Temporal-spatial interaction between reactive oxygen species and abscisic acid regulates rapid systemic acclimation in plants.

Being sessile organisms, plants evolved sophisticated acclimation mechanisms to cope with abiotic challenges in their environment. These are activated at the initial site of exposure to stress, as well as in systemic tissues that have not been subjected to stress (termed systemic acquired acclimation [SAA]). Although SAA is thought to play a key role in plant survival during stress, little is known about the signaling mechanisms underlying it. Here, we report that SAA in plants requires at least two different signals: an autopropagating wave of reactive oxygen species (ROS) that rapidly spreads from the initial site of exposure to the entire plant and a stress-specific signal that conveys abiotic stress specificity. We further demonstrate that SAA is stress specific and that a temporal-spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. In addition, we demonstrate that the rapid ROS signal is associated with the propagation of electric signals in Arabidopsis thaliana. Our findings unravel some of the basic signaling mechanisms underlying SAA in plants and reveal that signaling events and transcriptome and metabolome reprogramming of systemic tissues in response to abiotic stress occur at a much faster rate than previously envisioned.

Microvascular reactivity is altered early in patients with acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is associated with vascular endothelial dysfunction. The resultant microvascular reactivity can be assessed non-invasively using near-infrared spectroscopy (NIRS) and a vascular occlusion test (VOT) and changes have been correlated with severity of organ dysfunction and mortality in other critically ill populations. We used NIRS to study the presence of microcirculatory alterations in patients with ARDS. METHODS: We studied 27 healthy volunteers and 32 ARDS patients admitted to our intensive care department. NIRS measurements were performed within 24 h after diagnosis (Berlin definition). VOTs were performed by inflating an arm-cuff to a pressure greater than the systolic pressure for 3 min, followed by rapid deflation. The descending (Desc) and ascending (Asc) thenar muscle oxygen saturation (StO2) slopes were calculated. We compared data from volunteers with those from ARDS patients, from ARDS survivors and non-survivors, and from ARDS survivors who required <7 days ventilatory support (good evolution) with those who required >7 days support or died (poor evolution). RESULTS: ARDS patients had lower StO2 values [75(67-80) vs 79(76-81) %, p = 0.04] and Asc slopes [185(115-233) vs 258(216-306) %/min, p < 0.01] than healthy volunteers, but Desc slopes were similar. The Asc slope was lower in the patients with a poor evolution than in the other patients [121(90-209) vs 222(170-293) %/min, p < 0.01], and in the non-survivors than in the survivors [95(73-120) vs 212(165-252) %/min, p < 0.01]. CONCLUSIONS: In ARDS patients, microvascular reactivity is altered early, and the changes are directly related to the severity of the disease. The ascending slope is the best determinant of outcome.

Genetic association analysis of miRNA SNPs implicates MIR145 in breast cancer susceptibility.

BACKGROUND: MicroRNAs (miRNAs) are important small non-coding RNA molecules that regulate gene expression in cellular processes related to the pathogenesis of cancer. Genetic variation in miRNA genes could impact their synthesis and cellular effects and single nucleotide polymorphisms (SNPs) are one example of genetic variants studied in relation to breast cancer. Studies aimed at identifying miRNA SNPs (miR-SNPs) associated with breast malignancies could lead towards further understanding of the disease and to develop clinical applications for early diagnosis and treatment. METHODS: We genotyped a panel of 24 miR-SNPs using multiplex PCR and chip-based matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis in two Caucasian breast cancer case control populations (Primary population: 173 cases and 187 controls and secondary population: 679 cases and 301 controls). Association to breast cancer susceptibility was determined using chi-square (X (2) ) and odds ratio (OR) analysis. RESULTS: Statistical analysis showed six miR-SNPs to be non-polymorphic and twelve of our selected miR-SNPs to have no association with breast cancer risk. However, we were able to show association between rs353291 (located in MIR145) and the risk of developing breast cancer in two independent case control cohorts (p = 0.041 and p = 0.023). CONCLUSIONS: Our study is the first to report an association between a miR-SNP in MIR145 and breast cancer risk in individuals of Caucasian background. This finding requires further validation through genotyping of larger cohorts or in individuals of different ethnicities to determine the potential significance of this finding as well as studies aimed to determine functional significance.

Near infrared spectroscopy (NIRS) to assess the effects of local ischemic preconditioning in the muscle of healthy volunteers and critically ill patients.

Near-infrared spectroscopy (NIRS) permits non-invasive evaluation of tissue oxygen saturation (StO2). A vascular occlusion test (VOT) produces transient controlled ischemia similar to that used in ischemic preconditioning. We hypothesized that we could evaluate local responses to ischemic preconditioning by performing repeated VOTs and observing the changes in different NIRS VOT-derived variables. In healthy volunteers (n=20), four VOTs were performed at 30-min intervals on one day and, in a second group (n=21), two VOTs with time intervals of 5, 15 or 30min were performed on 3 separate days. Two cohorts of patients, one with circulatory shock (n=23) and a hemodynamically stable group (n=20), were also studied, repeating the VOT twice with a 5-min interval. In the 1-day volunteers, there was a median decrease of 15 (6-21)% in the Desc slope (StO2 decrease during VOT) after the second VOT, but no significant change in the Asc slope (StO2 increase after VOT). In the 3-day volunteers, the Desc slope also decreased, regardless of the time interval between VOTs. There was no overall decrease in the Desc slope in either patient cohort with repeated VOTs but there was marked individual patient variability. Patients in whom the Desc slope decreased had less organ dysfunction at admission, required less norepinephrine (0.00 vs 0.08mcg/kg/min, p=0.02), less frequently had sepsis (12 vs 50%, p=0.02) and had a lower mortality (6 vs 39%, p=0.03) compared to those in whom it did not decrease. Repeated NIRS VOT can non-invasively assess the local effects of ischemic preconditioning in the muscle.

Normobaric hyperoxia alters the microcirculation in healthy volunteers.

The use of high concentrations of inhaled oxygen has been associated with adverse effects but recent data suggest a potential therapeutic role of normobaric hyperoxia (NH) in sepsis and cerebral ischemia. Hyperoxia may induce vasoconstriction and alter endothelial function, so we evaluated its effects on the microcirculation in 40 healthy adult volunteers using side-stream dark field (SDF) video-microscopy on the sublingual area and near-infrared spectroscopy (NIRS) on the thenar eminence. In a first group of volunteers (n=18), measurements were taken every 30 min: at baseline in air, during NH (close to 100% oxygen via a non-rebreathing mask) and during recovery in air. In a second group (n=22), NIRS measurements were taken in NH or ambient air on two separate days to prevent any potential influence of repeated NIRS measurements. NH significantly decreased the proportion of perfused vessels (PPV) from 92% to 66%, perfused vessel density (PVD) from 11.0 to 7.3 vessels/mm, perfused small vessel density (PSVD) from 9.0 to 5.8 vessels/mm and microvascular flow index (MFI) from 2.8 to 2.0, and increased PPV heterogeneity from 7.5% to 30.4%. Thirty minutes after return to air, PPV, PVD, PSVD and MFI remained partially altered. During NH, NIRS descending slope and NIRS muscle oxygen consumption (VO2) decreased from 8.5 to 7.9%/s and 127 to 103 units, respectively, in the first group and from 10.7 to 9.4%/s and 150 to 115 units in the second group. NH, therefore, alters the microcirculation in healthy subjects, decreasing capillary perfusion and VO2 and increasing the heterogeneity of the perfusion.